First meta-analysis study of cholinesterase inhibition in experimental animals by organophosphate or carbamate insecticides under the influence of diphenhydramine

Background and Aim: Diphenhydramine is an H1-antihistamine that counteracts the toxic effects of organophosphate and carbamate insecticides that inhibit cholinesterase (ChE) activity. This meta-analysis aimed to investigate the effects of diphenhydramine on ChE inhibition induced by these insecticides in the plasma, erythrocytes, or whole brain of experimental animals. Materials and Methods: A data search was performed on erythrocyte, plasma, and brain ChE inhibition caused by organophosphate and carbamate insecticides in experimental animals (mice, rats, and chicks) treated with the antihistamine diphenhydramine in accordance with preferred reporting items for systematic reviews and meta-analysis, which was done by the two-group random-effects model meta-analysis. The meta-analysis included 18 records extracted from six studies that, appeared from 1996 to 2022. Results: Using the random-effects model, a two-group meta-analysis revealed that the combined effect size (ChE inhibition) was significantly more favorable in the control group than in the diphenhydramine intervention, as shown by a forest plot. The combined effect size (standardized mean difference) was 0.67, with a standard error of 0.3, a lower limit of 0.04, and an upper limit of 1.29 (p = 0.025). The heterogeneity was moderate, as I2 of the combined effect size was 74%, with a significant Cochrane Q-test result (Q = 65, p < 0.0001). Subgroup analysis indicated that, with brain ChE inhibition, the heterogeneity (I2) became 5%, which was lower than ChE inhibition in plasma (84%) and erythrocytes (78%). No publication bias was identified using the funnel plot and Egger’s test. Conclusion: This meta-analysis suggests that, in addition to its documented antidotal action against ChE-inhibiting insecticides, diphenhydramine can also reduce the extent of ChE inhibition, especially in the brain, which is the main site of toxicity of these insecticides. There is a need for additional studies to assess such enzyme inhibition in different parts of the brain.


Introduction
In veterinary clinical practice, public health, and agriculture, there is a widespread use of formulations of organophosphate (OP) or carbamate (CA) insecticides to control pests [1][2][3]. Such insecticides can result in poisoning by inhibiting the key enzyme cholinesterase (ChE) at the nerve terminals, leading to cholinergic overstimulation with subsequent effects on muscarinic, nicotinic, and central nervous systems and the production of a cascade of events potentially resulting in respiratory failure and death [4][5][6]. However, the inhibition of ChE activity by OP is irreversible, whereas that by CA is reversible [4,6].
Based on the properties of these insecticides in inhibiting ChE, ChE activities in blood, including those in serum or plasma cholinesterase (PChE) and erythrocytes cholinesterase (EChE), are usually measured as biomonitoring of the exposure to pesticides and to assess the status of poisoning [7,8].
Regarding the mechanism behind the antidotal action of diphenhydramine against OP or CA insecticide, its antimuscarinic and possibly antinicotinic properties have been highlighted [9,10,22,23]. However, there is no clear understanding of the interaction of diphenhydramine with antiChE insecticides at the level of the ChE. By examining OP-or CA-induced inhibition of ChE in the blood (PChE and EChE) or whole-brain cholinesterase (BChE) following in vivo animal experiments, it was reported that diphenhydramine did not affect the extent of ChE inhibition in rats [14,15] and mice [13], whereas the drug reduced the ChE inhibitory effects of OP and CA in chicks [20][21][22]. It was also reported that, in vitro, diphenhydramine partially reduced the inhibitory actions of the CA insecticide carbaryl on PChE and EChE in humans [30] and on PChE and BChE in chicks [22]. However, one study performed in mice indicated that PChE inhibition was increased by the combination of diphenhydramine and OP dichlorvos [31]. Meanwhile, diphenhydramine alone was reported to exhibit inhibitory effects on PChE and BChE in vivo in chicks [32]. These findings raised the possibility that diphenhydramine could weakly inhibit OP-induced ChE inhibition [21,22,30], as found with other weak ChE inhibitors [33,34].
Against this background, and because of the various, although conflicting, studies on ChE activities under the influence of diphenhydramine with OP or CA insecticides, this meta-analysis was established to investigate for the 1 st time the effects of antihistamine on ChE inhibition induced by OP (dichlorvos) or CA (carbaryl, methomyl) insecticides, as found in the literature.

Ethical approval
This is a meta-analysis, which was approved by the Reviewing Board, College of Pharmacy, University of Duhok, Iraq, and no live animals were used.

Study period and location
The study was conducted from January 2, 2022, to July 31, 2022, at the College of Pharmacy, University of Duhok, Iraq.

Search strategy
The data search was based on research articles and academic theses published from 1980 to July 31, 2022, on the combined effects of diphenhydramine with OP or CA insecticides in experimental animals (mice, rats, chicks), using PubMed, Google Scholars, Scopus (Science Direct), Directory of Open Access Journals, Semantic Scholar, Web of Science, My Research Assistant, and Iraqi Academic Scientific Journals databases. A manual search was performed on articles and academic theses from Iraqi universities because most of these resources do not appear in the above-mentioned databases. No language restriction was applied to the search strategy.

Selection criteria
As shown in Figure-1, 18 records were extracted from six studies on the inhibitory effects of OP or CA insecticides on PChE, EChE or BChE activities in experimental animals subjected to various treatment regimens with diphenhydramine. The records were selected in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) [35]. The selected papers included experimentally obtained values of blood or tissue ChE activities (mean, standard deviation, and standard error) under the in vivo influence of OP or CA insecticides with concurrent diphenhydramine (experimental intervention) or saline (control) treatment. The diphenhydramine treatments were administered either before or after the OP or CA insecticide dosing.

Inclusion and exclusion criteria
Studies included in the meta-analysis were those reporting effects of diphenhydramine on ChE activities in blood or tissue in experimental animals treated with OP or CA insecticide. Records were excluded if they were from studies using antihistamines other than diphenhydramine or did not report ChE activity in experimental animals. In vitro studies were also excluded from the study.

Data extraction
Data on ChE activities, as described in the selection criteria, were extracted from the texts, tables, or figures of the identified manuscripts. They reported ChE inhibition in experimental animals subjected to OP or CA insecticide treatments with or without diphenhydramine therapy. Some papers reported more than one subset of ChE inhibition data; such data were also included in the meta-analysis. We calculated the standard deviations from the corresponding standard errors depending on the data presented in the studies. When the numbers of experimental animals were presented as a range of minimum-maximum per group, the minimum number was chosen, to avoid increasing the favorable outcome toward the treatment effect. Any disagreement about data extraction was resolved by the authors of this paper through discussion.

Statistical analysis
A two-group meta-analysis was conducted in accordance with PRISMA guidelines using the random-effects model because of the expected sources of heterogeneity among different studies (Table-1) [14,15,[20][21][22]31]. The meta-analysis was performed using the software Meta-Essentials Version 1.5 (https://www.erim.eur.nl/research-support/meta-essentials/download/), which included a Available at www.veterinaryworld.org/Vol.16/January-2023/14.pdf  forest plot to calculate the effect size, weighted means with their 95% confidence intervals, a funnel plot, and subgroup analysis [36]. Along with visualizing the meta-analysis as a forest plot, a drapery plot was produced using Meta-Mar software (https://www. meta-mar.com/) as further supporting evidence, with proposed confidence intervals at p < 0.05 [37].

Publication bias
A funnel plot (size effect against standard error) was used to visually assess any publication bias, while Egger's statistical test was applied to objectively confirm the existence of such bias [40,41]. Figure-1 shows the PRISMA flow chart for selecting study records on blood or tissue (brain) Available at www.veterinaryworld.org/Vol.16/January-2023/14.pdf ChE inhibition caused by OP or CA insecticides in experimental animals treated with diphenhydramine. Initially, the search strategy retrieved 32 studies involving diphenhydramine use, either therapeutically in humans or animals or experimentally in animals suffering from OP or CA poisoning. The final list of selected records (n = 18) for the meta-analysis included those from five articles and one thesis ( Figure-1 and Table-1). They specifically involved inhibition of ChE activities in the blood or whole brain of experimental animals (mice, rats, and young chicks) under the influence of OP or CA insecticide with or without concomitant diphenhydramine therapy. These records were published between 1996 and 2022 (Table-1). The insecticides used in the selected studies were the OP dichlorvos in mice [31], rats [15], and chicks [20,21], or the CA methomyl in rats [14] or carbaryl in chicks [22]. The cumulative number of experimental animals with OP or CA insecticide intoxication in the selected studies was 103 for the diphenhydramine treatment and also 103 for the control one (Table-1). The enzyme sources were PChE, EChE, and BChE, comprising 94, 34, and 78 samples, respectively (Table-1).

Meta-analysis
Using the random-effects model, a two-group meta-analysis of these records revealed that the combined effect size (ChE inhibition) was significantly more favorable in the control group, as shown by the forest plot, indicating the value of diphenhydramine for treating OP or CA poisoning (Figure-2). This value was conferred via lower ChE inhibition than in the control group. The related statistics were as follows: combined effect size (standardized mean difference) 0.67, standard error 0.3, lower limit 0.04, upper limit 1.29, and Z value 2.24 with a two-tailed p-value of 0.025. The percentages of weights of individual records obtained from the forest plot were close to each other and ranged from 4.41% to 6.25% (Figure-2).

Heterogeneity analysis
We used Cochrane Q-test and the I 2 index to examine the heterogeneity among the studies of this random-effects model of meta-analysis (Figure-2). The heterogeneity I 2 index (with T 2 and T values of 1.04 and 1.02, respectively) of the combined effect size was 74%, indicating a moderate degree of heterogeneity, which was also confirmed to be statistically significant using the Cochrane Q-test (Q = 65, p < 0.0001).

Figure-2:
Forest plot of the records of comparison of cholinesterase inhibition in the plasma, erythrocytes or brain of experimental animals (mice, rats, and chicks) intoxicated with organophosphate or carbamate insecticides treated with diphenhydramine (left side of zero effect size) or with saline as untreated controls (right side of zero effect size).

Drapery plot
In addition to the forest plot, a drapery plot with proposed confidence intervals was applied to these data (using Meta-Mar software). The drapery plot (Figure-3) provided support for this meta-analysis based on the p-value functions of each record (confidence curve, Y axis = p values; X axis = effect size). Furthermore, Figure-3 shows meta-analysis estimates, and the shaded area depicts a prediction region for a single future study, suggesting the possibility of a shift of the point of effect size toward the diphenhydramine intervention.

Publication bias (funnel plot)
To examine whether there was publication bias, the funnel plot was visually examined. This indicated no publication bias, given the symmetrical distribution of the effect size points in a limited area of the plot (Figure-5). This conclusion was further objectively and statistically confirmed by Egger's test, the result of which was not significant (t = 0.59, p = 0.56).
This meta-analysis was conducted to resolve the potential beneficial effects of diphenhydramine regarding ChE inhibition. It demonstrated the beneficial effects of diphenhydramine in cases of poisoning by OP or CA insecticides in experimental animals at the level of ChE. This was shown by the combined effect size (high ChE inhibition) being significantly more favorable in the control-untreated (non-diphenhydramine) group; that is, lower ChE inhibition in the diphenhydramine intervention group (Figure-2). However, it should be stressed here that the activities of all three ChEs were significantly inhibited by OP or CA insecticides used in the experimental animals (Table-1). This was noticed in the studies regardless of concurrent diphenhydramine therapy, which was administered either before or after the intoxication by insecticides. In this context, it is impossible to draw definitive conclusions on the final clinical outcome of the extent of ChE inhibition after the diphenhydramine intervention, as the time points of blood or tissue sampling were within a few hours of intoxication. However, the clinical use of diphenhydramine in cases of OP or CA poisoning has been advocated in various animal species [9,[25][26][27][28][29] as well as in humans [23,24].
This meta-analysis suggested significant heterogeneity (albeit with a moderate I 2 index of 74%) in the results. Interestingly, subgroup analysis demonstrated no heterogeneity with BChE (I 2 = 5%), while PChE and EChE had high heterogeneity (I 2 = 84% and 78%, respectively). It is plausible that the heterogeneity was attributable to the inherent variations in ChE activities found in the plasma, erythrocytes, and brain [42][43][44]. BChE activity is most closely related to the actual clinical situation of OP or CA poisoning, which results in enzyme inhibition and subsequently produces the characteristic toxidrome of antiChE poisoning [4][5][6]42]. Furthermore, supporting the importance of BChE, subgroup analysis revealed that the weight of BChE inhibition in the analysis was 81%, compared with 12% and 7% for PChE and EChE, Figure-3: Drapery plot of the records of comparison of cholinesterase inhibition in the plasma, erythrocytes or brain of experimental animals (mice, rats, and chicks) intoxicated with organophosphate or carbamate insecticides treated with diphenhydramine or with saline as untreated controls. The plot shows p-value function (confidence curve) for individual records as well as meta-analysis estimates, and the shaded area depicts a prediction region for a single future study.
respectively (Figure-4). However, studies of BChE used in this meta-analysis were conducted on wholebrain homogenate; there is thus a need for detailed studies about the combined effects of diphenhydramine and OP or CA insecticides in different parts of the brain, which are involved in the insecticides' toxic outcomes [4][5][6]. Furthermore, all of the studies measured enzyme activities in the blood or the brain by a modified electrometric method [44], thus eliminating the factor of methodological variation that could influence the outcome measures [43]. However, other sources of heterogeneity could be the types of insecticides used, since they variably and differentially inhibit ChEs, and the species of experimental animals, since they inherently possess ChEs of various physiological activities in the blood or brain [43,44]. In support of our meta-analysis, which was conducted on blood and brain ChEs in vivo (mice, rats, and chicks), in vitro studies on human PChE and EChE [30] and chick PChE and BChE [21,22] suggested that diphenhydramine might partially protect the enzyme from additional inhibition induced by OP or CA insecticides. However, at the current stage, we cautiously draw such a conclusion, since the prediction of a future study by the drapery plot suggested the possibility of a shift of the point of effect size toward the diphenhydramine intervention. There is thus a need for additional studies on the effects of diphenhydramine following intoxication with various OP and CA insecticides in experimental animals.
Visual examination of the funnel plot together with results of the objective Egger's test indicated an absence  of publication bias in the outcome covering the studies under this meta-analysis. In the contour-enhanced funnel plot, there was a symmetrical distribution of the effect size points (Figure-5). This obviated the need for trim-and-fill analysis in the funnel plot [40,41].

Conclusion
This meta-analysis suggests that, in addition to its documented antidotal properties against poisoning with OP or CA insecticides, diphenhydramine could also reduce the extent of their ChE inhibition, especially in the brain, which is the main target of toxicity upon intoxication with these insecticides. There is a need for additional studies about the interaction of diphenhydramine with ChE-inhibiting insecticides in different parts of the brain, which are crucial in cases of poisoning with these insecticides.

Authors' Contributions
FKM: Conceptualized the study and drafted the manuscript. FKM, HMSG, AAM and HMR shared equally in the review and meta-analysis, provided comments, participated in writing the manuscript, and approved the final version of the manuscript.